The overall goal of this application covers the molecular, immunological, pharmacological, regulatory and functional aspects of the phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK, or protein kinase C) system in heart. The specific aims are as follows: (1). PL/Ca-PK, The enzyme. Identification, characterization of the catalytic and regulatory domains of the native enzyme (80 kDa) will be carried out using various methods, such a immunoreactivity with antiserum to the native enzyme by ELISA or immunoblots, and labeling of the ATP-, phosphatidylserine- or Ca2+-binding sites. Poly- and/or monoclonal antibodies to the purified domains will be developed and used for the immunocytochemical localization of the domains, compared with that of the native enzyme, in myocardium and cultured myocytes influenced by drug interventions or pathophysiology. The gene for the enzyme will be cloned using the polyclonal antibodies against the native enzyme already developed in this lab. (2). Substrates for PL/Ca-PK: The substrate determinants for the enzyme will be further probed using synthetic peptides of myelin basic protein (MBP) having amino acid sequence segments around the phosphorylated serine-115. The phosphorylation sites in cardiac troponin I and troponin T as well as the amino acid sequence around the phosphorylation sites will be determined. Functional significance of troponin phosphorylation will be studied by investigating any changes in the activity and Ca2+-sensitivity of actomyosin ATPase. (3). Regulation of PL/Ca-PK system: The inhibitor peptides (analogs of MBP substrate peptides) will be further studied by modifying their amino acid residues or peptide lengths. The newly-identified cardiac endogenous inhibitor protein will be purified, and its active site(s) determined. Peptides having amino acid sequence segments around the active sites will be synthesized and tested for its inhibitory activity. The mechanisms of action of the inhibitor peptides (derived from MBP, troponin and endogenous inhibitor) will be examined, and compared with those of other known inhibitors (such as cardiotoxin I). It is hoped that the present proposal would provide new knowledge concerning regulation of cardiac function at the level of protein phosphorylation catalyzed by this major, multifunctional protein kinase.